Method of preparing a modified corn steep liqour product

ABSTRACT

A method of preparing a modified corn steep liquor product from a corn steep liquor which includes suspended solids, high molecular weight compounds, phytic acid, and metal values includes the steps of: subjecting the corn steep liquor to ultrafiltration to remove the suspended solids and high molecular weight compounds; treating the product of step (a) with a phytase enzyme to convert the phytic acid to inositol-mono-phosphate; treating the product of step (b) with an acid phosphatase enzyme to convert the inositol-mono-phosphate to myo-inositol; adjusting the pH of the product of step (c) to from 7 to 9 inclusive using a soluble base which causes most or all of the Mg values to precipitate, and removing the precipitate; and contacting the product of step (d) with a strong acid cation chromatographic resin to produce a first fraction containing most of the remaining metal values and a second fraction containing most of the myo-inositol, the second fraction being the modified corn steep liquor product.

BACKGROUND OF THE INVENTION

[0001] THIS invention relates to a method of preparing a modified cornsteep liquor product which is enriched in myo-inositol, from a cornsteep liquor.

[0002] Myo-inositol (also known as inositol, muscle sugar, andhexahydroxycyclohexane) is an important growth factor considered by someas part of the B vitamin group (Szmant, “Industrial utilization ofrenewable resources”, Technomic Publication Company, Lancaster, Pa.,1986). It is found in plants both in the free form (in soya and sugarcane for instance) and also as a hexaphosphate, phytin, (in corn forinstance) and also as a phosphatidyl derivative, which is a component ofvegetable lecithins.

[0003] The main applications of myo-inositol are in aquaculture, petfood, animal feed and in the developing markets in health and nutrition.

[0004] Corn steep liquor is the traditional industrial source ofmyo-inositol (e.g Artz and Hach, “Process for refining crude inositolcontaining solutions and for recovering inositol therefrom”, U.S. Pat.No. 2,615,053, Oct. 21, 1952). In this process, phytin is firstprecipitated as a calcium salt and then hydrolysed with a strong acid.The production of myo-inositol in this fashion was stopped in the USA inabout 1987 because of high production costs. Thus, supplies ofmyo-inositol now come from China (where it is made from corn steepliquor) and also from Japan (where it is made from rice bran).

[0005] U.S. Pat. No. 3,270,064 to Toyo Koatsu Industries Inc, describesan improvement to the chemical precipitation and hydrolysis process byusing a soluble base to raise the pH (e.g ammonia or caustic) andprecipitate the phytin. This is followed by hydrolysis under heat andpressure in the presence of an alkaline earth metal carbonate such asmagnesium carbonate. Benefits claimed are elimination of complexingreactions and improved purity of the final product. Chemical consumptionis still high and the use of caustic or ammonia detracts from the valueof the remaining material, as it is not possible to use it as feed.

[0006] Because of the chemicals involved in the precipitation andhydrolysis of phytin, any source of the free form of myo-inositol willbe at an advantage, provided an efficient way can be found to separatethe myo-inositol from the other components. To this effect two processeshave been patented, viz. U.S. Pat. No. 5,096,594 to Rabinowitz and U.S.Pat. No. 4,482,761 to Chao and Sherman. One process is based on the useof cation exchange resins in Ca²⁺ or Pb²⁺ form with aqueous dimethylsulfoxide as the mobile phase and the other is based on zeolitemolecular sieves. The former process has the disadvantages of using anorganic solvent and the use of lead in a food product is entirelyunacceptable. The latter process is not very efficient.

[0007] Myo-inositol cannot be efficiently separated from monosaccharides(glucose and particularly fructose) on cation exchange resins inmonovalent (Na⁺ or K⁺) or divalent (Ca²⁺) form. The use of strong baseanion resins to separate myo-inositol from sugar steams has beendemonstrated as a feasible option for molasses type streams from thesugar industry (Saska, “Industrial production of inositol: A by-productfrom the cane molasses desugarization process” 1997, Int. Sugar Jnl.,(99): 480-484).

[0008] Corn steep liquor cannot be used directly as a feed for sensitivefeed applications such as small animals and especially weaning piglets,as the high level of potassium and other salts promote scours and otherdigestive disorders. Nevertheless, corn steep liquor is a high qualityprotein source, rich in highly available soluble protein and is ofinterest in specialist feed applications, provided the problematiccomponents can be separated therefrom.

SUMMARY OF THE INVENTION

[0009] According to the invention there is provided a method ofpreparing a modified corn steep liquor product from a corn steep liquorwhich includes suspended solids, high molecular weight compounds, phyticacid, and metal values (Mg, Ca, Fe, Mn, Zn, Na, K), including the stepsof:

[0010] (a) subjecting the corn steep liquor to ultrafiltration to removethe suspended solids and high molecular weight compounds;

[0011] (b) treating the product of step (a) with a phytase enzyme toconvert the phytic acid to inositol-mono-phosphate;

[0012] (c) treating the product of step (b) with an acid phosphataseenzyme to convert the inositol-mono-phosphate to myo-inositol;

[0013] (d) adjusting the pH of the product of step (c) to from 7 to 9inclusive using a soluble base which causes most or all of the Mg valuesto precipitate, and removing the precipitate; and

[0014] (e) contacting the product of step (d) with a strong acid cationchromatographic resin to produce a first fraction containing most of theremaining metal values, and a second fraction containing most of themyo-inositol, the second fraction being the modified corn steep liquorproduct.

[0015] In step (a), the ultrafiltration is preferably carried out in therange of from 10 000 kD to 100 00 kD inclusive, preferably in the rangeof from 20 000 kD to 40 000 kD inclusive.

[0016] The suspended solids and high molecular weight compounds areremoved as they lead to fouling of the resin in step (e).

[0017] Any suitable membrane material may be used in theultrafiltration, for example PVDF (polyvinylidene di-fluoride).

[0018] In step (b), the phytase enzyme may be used in free form or maybe immobilized onto any suitable carrier.

[0019] In step (c) the acid phosphatase enzyme may be used in free formor may be immobilized onto a suitable carrier.

[0020] Steps (b) and (c) can be combined into one step, i.e are carriedout simultaneously.

[0021] In step (d), the pH is preferably adjusted to a value of from 7,5to 8,5 inclusive.

[0022] The preferred base is ammonia as this causes precipitation ofstruvite which is NH₄MgPO₄.6H₂O, thereby removing substantially all ofthe Mg values The Mg values would otherwise interfere with the resin instep (e).

[0023] The recovered struvite is a high value slow release fertiliser.

[0024] Another base which may be used is potassium hydroxide, which alsoleads to precipitation of the Mg values present.

[0025] In step (e), the first fraction, which may be termed the ashfraction, contains lactate, sulphate, phosphate and chloride values, aswell as the remaining metal values.

[0026] The ash fraction may also be dried to produce a potassium richfertilizer. Alternatively, potassium salts may be crystallised from thesolution and the remaining solution used as a liquid fertilizer.

[0027] The second fraction contains the myo-inositol, as well as otherdesirable components such as proteins and sugars.

[0028] After step (e), the modified corn steep liquor product may beevaporated and dried to produce a dry product, enriched in myo-inositol.

BRIEF DESCRIPTION OF THE DRAWINGS

[0029]FIG. 1 is a chromatogram from the Experimental Work; and

[0030]FIG. 2 is a flow diagram illustrating a continuous simulatedmoving bed from the Experimental Work.

DESCRIPTION OF EMBODIMENTS

[0031] The crux of the invention is a method of preparing a modifiedcorn steep liquor product from a corn steep liquor which includessuspended solids, high molecular weight compounds, phytic acid and metalvalues generally in the form of metal ions, the product being enrichedin myo-inositol.

[0032] The invention will now be described in more detail with referenceto the following experimental work:

Experimental Work

[0033] Step (a)—Ultrafiltration

[0034] Crude corn steep liquor (CSL) from a corn wet mill was diluted to30% (m/m) solids and fed to a Niro ultrafiltration plant with thefollowing operating conditions:

[0035] Advanced Mambrane Technology Inc. (AMT) spiral wound PVDFmembranes of 30 000 kD,

[0036] Cross flow velocity 2-4 m/s using 80 mils spacers (2 mm) Pressuredrop 1 Bar,

[0037] Trans-membrane pressure (TMP) of 4-5 Bar,

[0038] Concentration ratio (retentate/feed) of 0,5.

[0039] Turbidity of permeate (suspended solids) less than 10 NTU(nephelometric turbidity units) as measured by nephelometer.

[0040] Feed pH of 4,0,

[0041] Temperature of 40° C.

[0042] The result is a product from which suspended solids and highmolecular weight compounds, which would lead to fouling of the resin instep (e), are removed.

[0043] Step (b)—Phytase Treatment

[0044] The myo-inositol in the clarified CSL from step (a) isessentially present as phytic acid or as a salt thereof. Being aphosphate containing product, this product would follow the ash fractionin any separation, and thus the phytic acid must be converted to freemyo-inositol before separation of the ash fraction. This is achieved byenzymatic hydrolysis of the phytic acid to inositol-mono-phosphate usinga phytase enzyme. The treatment conditions are as follows:

[0045] Enzyme used: BASF Natuphos as described in BASF product brochurein free form,

[0046] Enzyme dosage: 500 FTU (phytase activity units) per kg CSL (as30% solids),

[0047] Optimum pH: 3,0 to 4,0 to ensure adequate phytase activity andalso shelf life of the CSL,

[0048] Temperature of 40° C.,

[0049] Residence time: 12 hours.

[0050] The resulting product contains inositol-mono-phosphate. Ananalysis of a typical product from step (b) is as follows: ComponentUnits Value General analyses Density Kg/m³ 1150 pH 3.0 Dry matter (Brix)% (m/m) 31.0 Organic components Glucose % m/m 4.21 Frutose % m/m 1.68D-lactate % m/m 5.05 L-lactate % m/m 5.61 Phytic Acid % m/m <1Inositol-mono-phosphate % m/m 5.61 Amino Acids Threonine % m/m 0.45Methionine % m/m 0.45 Valine % m/m 1.57 Alanine % m/m 2.69 Glycine % m/m0.34 Aspartate % m/m 0.17 Glutamate % m/m 0.59 Serine % m/m 0.50Tyrosine % m/m 0.28 Arginine % m/m 0.76 Iso-leucine % m/m 0.45 Leucine %m/m 1.43 Histidine % m/m 0.14 Cysteine % m/m not measured Phenylalanine% m/m 0.84 Protein composition (dry basis) Total free amino acids %(m/m) 10.0 TKN protein % (m/m) 42.0 Minerals Mg ppm (m/m) 14516 Ca ppm(m/m) 645 Fe ppm (m/m) 210 Mn ppm (m/m) 113 Zn ppm (m/m) 226 Na ppm(m/m) 684 K % m/m 6.45 Chlorides % m/m 0.98 Free phosphates % m/m 1.21Sulphates % m/m 7.43

[0051] Step (C)—Acid Phosphatase Treatment

[0052] An acid phosphatase enzyme is used to hydrolyse theinostol-mono-phosphate containing a single phosphate group to freemyo-inositol and the free phosphate. The acid phosphatase treatmentconditions are as follows:

[0053] Enzyme used: Sumizyme PM-L from the Shin Nihon Company (activity3000 u/g).

[0054] Dosage: 1 unit of activity per g of CSL (as 30% solids),

[0055] Temperature of 65° C. and pH of 4,0 to 4,5,

[0056] Residence time; 24 hours,

[0057] Conversion achieved: >98% as determined by inositol and IMPmeasurement by HPLC.

[0058] Step (d)—pH Adjustment and Removal of Mg Values

[0059] It is desirable to conduct the chromatographic resin separationstep (Step (e)) without any divalent cations being present. It is wellknown that these cations have a negative impact on the resolution of thechromatographic separation.

[0060] Thus, to avoid this problem, the product of step (c) has its pHadjusted to a value between 7,5 and 8,5 using ammonia, to causeprecipitation of struvite.

[0061] The precipitate was collected and subjected to an XRF analysiswhich confirmed the material as being struvite. Analysis of N. Mg andPO₄ using wet chemical assays confirmed the presence of these compoundsin the exact stoichiometric quantities to be expected for pure struvite.It was also confirmed that there was very little K (±0,3%) and no Na,Cl, SO₄ or heavy metals present and that the purity was in excess of99%.

[0062] Struvite finds application as a slow release fertiliser.

[0063] The clear filtrate was also analysed and it was confirmed thatthe Mg values were essentially completely removed. This product was thenpassed on to the next step.

[0064] Step (e)—Chromatographic Separation

[0065] The separation was conducted in a single column with thefollowing operating conditions:

[0066] Column: 100 cm length with 1 cm diameter,

[0067] Resin: Rohm and Haas CR1320 chromatographic resin,

[0068] Temperature: 60° C.,

[0069] Resin volume: 78,5 ml,

[0070] Injection volume: 0,01 BV,

[0071] Flow rate: 3 ml/min of water (2,25 BV/h or roughly 2,3 m/hvelocity),

[0072] Detector: On line refractive index (Waters 400).

[0073]FIG. 1 is a chromatogram which shows the separation achieved asfollows: Void volume: 0.32 BV Fraction 1 (raffinate or ash) peak 0.40 BVFraction 2 (myo-inositol rich) peak 0.58 BN

[0074] The peak marked 1 is the ash fraction peak, and the peak marked 2is the myo-inositol rich fraction peak.

[0075] HPLC analyses indicated that the ash fraction contained thelactate, sulphate, phosphate and chloride values and all the cations.

[0076] Dry matter determinations indicated that the total solids of theCSL was split equally between fractions 1 and 2, with all themyo-inositol reporting to fraction 2. The concentration of myo-inositolwas 10% on a dry matter basis.

[0077] In order to optimise the chromatographic separation it isdesirable to operate a continuous device such as a simulated moving bed(SMB). In this way it is possible to optimise the eluent and feed flowsto achieve the best separation with minimum dilution, whilst maintainingsteady operation by effectively maintaining the separation peaks in astationary position. This means that the two fractions can be extractedfrom fixed ports as is illustrated in FIG. 2 which is described in moredetail below.

[0078] The design flowrates as indicated were determined from the batchchromatogram (pulse test) discussed above, using standard design andscale-up calculations as used by Advanced Separation Technologies (adivision of Calgon Carbon) to design separations based on their Iseprotating carousel system. The design criteria are as follows: Resinvolume:  2.2 litres in total (220 ml per column) Step time:   4 minResin flow:   55 ml/min Linear velocity:  7.5 m/h (safe value for resinof 320 micron) Bed depth: 0.45 m Feed rate:  0.1 BV/h Slow Product:Fraction 2 of Figure 2 being the myo-inositol enriched fraction FastProduct: Fraction 1 of Figure 2 being the ash containing fraction.

[0079] Referring to FIG. 2, there is shown a pilot setup for continuousseparation of myo-inositol enriched CSL.

[0080] The product of step (d) is fed into port 5 of the apparatus via aline 12, at a feed rate of 4 ml/min. Water is fed into port 1 of theapparatus via a line 12, at a feed rate of 27,5 ml/min.

[0081] Fraction 1 is removed from the apparatus at port 9 via a line 16,at a rate of 9,35 ml/min.

[0082] Fraction 2 is removed from the apparatus at port 2 via a line 18at a rate of 22,15 ml/min.

[0083] Product from port 10 is recycled via a line 20 to the water inletline 14. The numbers 1 to 10 in the drawing refer to stationary ports.Each of the ten columns containing the resin in the apparatus moves fromport to port with a step time of 4 minutes. At each step, each columnmoves to the next port, and in this way continuous countercurrent resinflow is achieved. The resin flow is 220 ml resin/4 min, or 55 ml/min.

[0084] Crystallisation Of Potassium Salts from the First Fraction

[0085] A small sub-sample of the concentrated first fraction was furtherconcentrated under vacuum in a rotary laboratory evaporator in order toinvestigate the feasibility of crystallizing potassium salts. Fineneedle like crystals of some 50×300 microns in size were produced and afew grams recovered with a laboratory centrifuge. The crystals weredetermined by powder X-ray diffraction analysis to be primarily apotassium hydrogen phosphate KH₂PO₄ (arcanite), with a smaller amount ofpotassium sulfate K₂SO₄.

1 A method of preparing a modified corn steep liquor product from a cornsteep liquor which includes suspended solids, high molecular weightcompounds, phytic acid, and metal values including the steps of: (a)subjecting the corn steep liquor to ultrafiltration to remove thesuspended solids and high molecular weight compounds; (b) treating theproduct of step (a) with a phytase enzyme to convert the phytic acid toinositol-mono-phosphate; (c) treating the product of step (b) with anacid phosphatase enzyme to convert the inositol-mono-phosphate tomyo-inositol; (d) adjusting the pH of the product of step (c) to from 7to 9 inclusive using a soluble base which causes most or all of the Mgvalues to precipitate, and removing the precipitate; and (e) contactingthe product of step (d) with a strong acid cation chromatographic resinto produce a first fraction containing most of the remaining metalvalues, and a second fraction containing most of the myo-inositol, thesecond fraction being the modified corn steep liquor product. 2 A methodaccording to claim 1 wherein in step (a) the ultrafiltration is carriedout in the range of from 10 000 kD to 100 000 kD inclusive. 3 A methodaccording to claim 2 wherein in step (a) the ultrafiltration is carriedout in the range of from 20 000 kD to 40 000 kD inclusive. 4 A methodaccording to any one of claims 1 to 3 wherein in step (a) theultrafiltration is carried out using a polyvinylidene di-fluoridemembrane material. 5 A method according to any one of claims 1 to 4wherein in step (b) the phytase enzyme is used in free form orimmobilized onto a carrier. 6 A method according to any one of claims 1to 5 wherein in step (c) the acid phosphatase enzyme is used in freeform or immobilized on a carrier. 7 A method according to any one ofclaims 1 to 6 wherein steps (b) and (c) are carried out simultaneously.8 A method according to any one of claims 1 to 7 wherein in step (d) thepH is adjusted to a value of from 7.5 to 8.5 inclusive. 9 A methodaccording to any one of claims 1 to 8 wherein in step (d) the solublebase is ammonia. 10 A method according to any one of claims 1 to 8wherein in step (d) the soluble base is potassium hydroxide. 11 A methodaccording to any one of claims 1 to 10 wherein after step (e) themodified corn steep liquor product is evaporated and dried to produce adry produce enriched in myo-inositol. 12 A method according to any oneof claims 1 to 11 wherein after step (e) potassium salts arecrystallised from the first fraction.